Toll-like receptors (TLRs) provide a mechanism for host defense by activating innate immune responses. Activated TLRs [e.g., by bacterial lipopolysaccharide (LPS)] dimerize, and interact with adaptor proteins through their cytosolic TIR domains to trigger a signaling cascade that ultimately leads to the expression of proteins involved in pro-inflammatory responses. One such adaptor protein is the TIR domain-containing adaptor protein (TIRAP; also known as MAL), which contains an N-terminal phosphatidylinositol 4,5- bisphosphate (PtdIns(4,5)P2)-binding region that is required for plasma membrane targeting and a C-terminal TIR domain, which mediates myeloid differentiation primary response gene 88 (MyD88) association. Upon ligand binding, the LPS-binding protein TLR4 is proposed to be recruited to PtdIns(4,5)P2-rich regions where TIRAP resides. At these sites, TIRAP recruits MyD88 to the plasma membrane via TIR-TIR domain interactions; thus TIRAP bridges MyD88 binding to activated TLR4. PtdIns(4,5)P2-mediated recruitment of TIRAP is considered to be the earliest cellular event required for TLR-mediated signaling and, consequently, it has been proposed that TIRAP defines the signaling sites at PtdIns(4,5)P2-rich membrane regions. Therefore, details of how TIRAP interacts in PtdIns(4,5)P2-rich membrane sites are crucial to understanding how the protein triggers downstream signaling upon microbial detection. A conserved stretch at the N-terminus of TIRAP (amino acids 15-35) has been shown to be sufficient to target the plasma membrane. Our preliminary data indicates that this region, which we name the PtdIns(4,5)P2 binding motif (PBM), folds in dodecylphosphocholine micelles, and binds PtdIns(4,5)P2. The solution structures of the micelle-associated and PtdIns(4,5)P2:micelle-bound states of TIRAP PBM will be solved and compared to precisely map the PtdIns(4,5)P2 binding site and define the structural basis of multi-step membrane insertion. Kinetics of the interactions of TIRAP PBM and deficient PtdIns(4,5)P2-binding mutants will be defined using NMR and surface plasmon resonance detection. The depth and angle of membrane penetration of TIRAP PBM will be elucidated with paramagnetic spin labels to better understand how binding influences membrane curvature. Given that TIRAP weakly binds to other acidic phospholipids, the contribution of these molecules in TIRAP's membrane insertion will be determined. We hypothesize that TIRAP membrane binding is regulated by the PtdIns(4,5)P2 head group, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which accumulates in the presence of extracellular LPS. The kinetics of TIRAP's Ins(1,4,5)P3 association will be measured and compared with those calculated for PtdIns(4,5)P2. Thus, these studies will provide a basis for understanding the mechanism and regulation of TIRAP's membrane targeting, which can be used for structure-based design of high affinity specific phosphoinositide-binding modules that ultimately contribute to pro-inflammatory responses.